Weeks 14 & 15
Thank you Samantha for submitting - Dr. B121714 Virtual Screening:
Figure 7: GOLD binding poses of the top 3 scoring ligands from the NIH clinical collection library (SAM001246783, SAM001246577, and SAM001246653). Poses are shown in PyMOL with 3N28 shown as a surface, and the ligands shown as sticks and colored by element with cyan carbons. Hydrogen bonds between the ligands and active site are represented by black dashes lines.
Figure 6: Best ranking table for the In House compounds library showing GOLD scores and Lipinski’s Rule data.
Figure 5: Best ranking table for Chembridge 306 library showing GOLD scores and Lipinski’s Rule data.
Figure 4: Best ranking table for NIH clinical collection library showing GOLD scores and Lipinski’s Rule data.
Analysis:
The vcPSP protein was screened against the NIH clinical collection, the Chembridge 306 library, and the library of in house compounds. Although these are small libraries, a few ligands had high binding scores. However, the two highest scoring ligands did not satisfy Lipinski's rule. The binding poses for the top ligands were analyzed in PyMol. It appears that GOLD did dock ligands in the active site of vcPSP properly. The next step will be to continue virtual screening by screening larger libraries.
Control Ligand Docking:
Figure 3: Best ranking table with GOLD scores and ranking for positive and negative control docking.
Figure 2: Molprobity results for the crystal structure of vcPSP (PDB ID: 3N28)
Figure 1: Active site of 3N28- Protein is shown as a surface and colored by element with green carbons. The active site is colored by element with cyan carbons. The ligand SEP is shown as sticks.
Analysis:
In preparation for virtual screening the active site of the vcPSP protein was defined by aligning it with a homologous protein (PDB ID: 1L7P), extracting the ligand, SEP, from that structure, and then defining the active site around this ligand. The protein was then prepared for virtual screening with MolProbity, and later with Hermes. First, a library of positive and negative control ligands was screened. Based on the GOLD docking scores for these ligands, it appears that the ligands selected were not very good controls. Many of the negative controls scored higher than the positive controls. This could be because the controls chosen were not very good, or it could be due to errors in the way that GOLD docked the ligands.
1242014- Outstanding work
Weeks 11, 12 & 13
Cloning Trial #3:
Figure 22: Plate C from cloning trial 3 showing growth of DH5-α transformed with vcPSP after about 16 hours in the 37 degree incubator. The cells were plated onto LB agar with kanamycin and sucrose.
Figure 21: Plate B from cloning trial 3 showing growth of DH5-α transformed with vcPSP after about 16 hours in the 37 degree incubator. The cells were plated onto LB agar with kanamycin and sucrose.
Figure 20: Plate A from cloning trial 3 showing growth of DH5-α transformed with vcPSP after about 16 hours in the 37 degree incubator. The cells were plated onto LB agar with kanamycin and sucrose.
Analysis:
There was some growth of colonies on the plates, although there also appears to be some contamination. The next step is to make a master plate and grow cultures overnight.
RE Digest:
Figure 19: Agarose gel results for restriction enzyme digest of pNIC cut with BsaI. Lane 2 contains the 1 Kb ladder. Lane 4 contains sample A of the digested pNIC, and lane 5 contains sample B of the digested pNIC.
Figure 17: Nanodrop spectrophotometry reading for digested pNIC sample A, trial 1.
Analysis:
The pNIC was successfully cut with BsaI restriction enzyme. Then it was cleaned using PCR clean-up to remove contamination.
Cloning, Trials 1 & 2:
Figure 16: Growth of DH5-α transformed with vcPSP after 2 days in the 37 degree incubator. The cells were plated onto LB agar with kanamycin and sucrose.
Figure 15: Growth of DH5-α transformed with vcPSP after 2 days in the 37 degree incubator. The cells were plated onto LB agar with kanamycin and sucrose.
Analysis:
Both of these cloning trials were unsuccessful as shown by the lack of colonies growing on the plates. There could have been errors making the pNIC accepting vector or with the transformation.
Secondary PCR and PCR Squared:
Figure 14: Agarose gel results for PCR2 using my secondary PCR sample trial 3. Lane 2 contains the 1 Kb ladder. Lanes 4-7 contain the PCR2 samples or vcPSP.
Figure 13: Agarose gel results for PCR2 using my secondary PCR sample trial 2. Lane 2 contains the 1 Kb ladder. Lanes 4-7 contain the PCR2 samples or vcPSP.
Figure 12: Agarose gel results for PCR2 using my secondary PCR sample. Lane 2 contains the 1 Kb ladder. Lanes 4-7 contain the PCR2 samples or vcPSP.
Figure 11: Agarose gel results for secondary PCR. Lane 2 contains the 1 Kb DNA ladder. Lane 4 contains my secondary PCR sample for vcPSP. Lane 9 contains James’s secondary PCR sample.
Analysis:
Secondary PCR was conducted so that more PCR squared could be made in preparation for cloning. The first to attempts at PCR squared were unsuccessful, likely due to errors with PCR technique.
RE Digest:
Figure 10: Agarose gel results for restriction enzyme digest of pNIC cut with BsaI. Lane 2 contains the 1 Kb ladder. Lane 4 contains sample A of the digested pNIC, and lane 6 contains sample B of the digested pNIC.
Analysis:
pNIC was sucessfully cut with BsaI as shown by the gel. The sacB gene was removed. The pNIC sample is know ready to use in cloning.
PCR Squared:
Figure 5: Nanodrop spectrophotometry reading for PCR clean up sample B, trial 2.
Figure 4: Nanodrop spectrophotometry reading for PCR clean up sample B, trial 1.
Figure 3: Nanodrop spectrophotometry reading for PCR clean up sample A, trial 2.
Figure 2: Nanodrop spectrophotometry reading for PCR clean up sample A, trial 1.
Figure 1: Agarose gel results for PCR2 using Avery’s secondary PCR sample 1. Lane 2 contains the 1 Kb ladder. Lanes 4-7 contain the PCR2 samples for vcPSP.
Analysis:
PCR squared was conducted to amplify the vcPSP gene. Now, enough of the gene has been made to do the cloning procedure.
1162014- Nice work Weeks 8,9 & 10
PCR Squared:
Figure 11: Agarose gel results for trial 2 of PCR squared using Avery’s secondary PCR sample #1. Lane 2 contains the 1 kb DNA ladder. Lanes 4-7 contain the 4 PCR squared samples for vcPSP.
Figure 10: Agarose gel results for trial 1 of PCR squared using Avery’s secondary PCR sample #2. Lane 2 contains the 1 kb DNA ladder. Lanes 3-6 contain the 4 PCR squared samples for vcPSP. Lane 7 contains James’s primary PCR sample.
Analysis: The purpose of PCR squared was to amplify the gene produced by secondary PCR. I used two different secondary samples for PCR squared, and only one of them was sucessful. The next step will be to redo PCR squared and PCR clean up or possibly gel extraction. After completing PCR my gene will be ready for cloning.
Secondary PCR:
Figure 9: Gel Results of trial 1 for primary PCR using Avery’s oligo mix. In lane 3 is the 1 Kb DNA ladder. The primary PCR sample for vcPSP is in lane 5.
Figure 8: Agarose Gel Results of trial 2 for primary PCR of oligo mix #2 for vcPSP. In lane 2 is the 1 Kb DNA ladder. The primary PCR sample for vcPSP is in lane 4.
Figure 7: Agarose Gel Results of secondary PCR temperature gradient number 2 for vcPSP. In lanes 1 and 2 is the 1 Kb DNA ladder. Lanes 3-10 contain the 8 secondary PCR samples from the temperature gradient.
Figure 6: Gel Results of trial 1 for primary PCR of oligo mix #2 for vcPSP.. In lane 2 is the 1 Kb DNA ladder. The primary PCR sample for vcPSP is in lane 4.
Figure 5: Gel Results of secondary PCR temperature gradient number 1 for vcPSP. In lane 2 is the 1 Kb DNA ladder. Lanes 3-10 contain the 8 secondary PCR samples from the temperature gradient.
Figure 4: Gel Results of trial 4 of secondary PCR for vcPSP. In lanes 2 and 5 is the 1 Kb DNA ladder. My secondary PCR sample for vcPSP is in lane 7.
Figure 3: Gel Results of trial 3 for secondary PCR for vcPSP. In lane 7 is the 1 Kb DNA ladder. My secondary PCR sample for vcPSP is in lane 9.
Figure 2: Gel Results of trial 2 for secondary PCR for vcPSP. In lane 2 is the 1 Kb DNA ladder. Lane 4 contains Keenan's secondary sample and lane 6 contains Avery's secondary PCR sample. My secondary PCR sample is in lane 8.
Figure 1: Gel Results of trial 1 for secondary PCR for vcPSP. In lanes 2 and 7 is the 1 Kb DNA ladder. The secondary PCR sample for vcPSP is in lane 8.
Analysis:
Secondary PCR was attempted using many different thermocycling conditions. However, none of the conditions used worked for secondary PCR. This could have been because the ideal conditions for my protein were not found, or it may have been caused by problems with PCR technique or experimental error. Avery's secondary sample was then used to move on to PCR squared.
9232014- Keep up the good work
Weeks 5,6 & 7
Primary PCR: Trial 3:
Figure 6: Gel results for trial 3 of Primary PCR for oligo mix for vcPSP. Lane 2 contains the 1 Kb DNA ladder, lane 3 contains my primary PCR sample for vcPSP, lanes 4 and 5 contain Alberto's primary and secondary PCR samples, and lane 6 contains Justin's restriction enzyme digest. Lane 9 contains the 100bp ladder.
Trial 2: Figure 5: Gel electrophoresis results for trial 2 of primary PCR for vcPSP oligo mix. Lane 2 contains the 1Kb DNA ladder, lane 3 contains my primary PCR sample with no visible results, and lane 4 contains Cidia's secondary PCR sample.
Trial 1:
Figure 4: Gel results for primary PCR of vcPSP oligo mix. Lane 2 contains the 1 kB ladder and lane 3 contains the PCR sample showing no visible bands.
Analysis:
The first 2 trials for my primary PCR were unsuccessful and nothing was seen on the gels for those trials. The third trial of primary PCR was successful, and a smear can clearly be seen on the gel. For trial one the NEB recommended guidelines were used for PCR, and for trial 2 the other guidelines were used. For the successful PCR the annealing time was changed from 10 to 20 seconds. This likely allowed more time for the oligos to successfully attach to one another.
Midiprep:
Figure 3: Nanodrop image for Midi Prep sample measured at 260 nm on the DNA-50 setting showing a 40.7 ng/uL yield for pNIC plasmid.
Figure 2: Nanodrop image for Midi Prep sample measured at 260 nm on the DNA-50 setting showing a 40.6 ng/uL yield for pNIC plasmid.
Analysis:
The purpose of Midi Prep was to isolate the pNIC plasmid that we grew from the cell pellet so that only the plasmid DNA remained in solution. Then the sample was nanodropped to determine the concentration of the plasmid. The average yield for my midiprep sample was 40.65 ng/uL, which is approximately what would be expected for pNIC because it is a low copy plasmid.
PCR- pGBR22:
Figure 1: Agarose gel results of PCR sample of purple protein coding sequence from pGBR22. Lane 2 is the 1 kb DNA ladder, lane 3 contains sample A with 0.3 ng plasmid, lane 4 contains sample B with 3 ng , lane 5 contains sample C with 30 ng and lane 6 contains the control sample D containing no DNA.
Analysis:
The PCR of the purple protein coding sequence of pGBR22 was successful The bands in lanes 3-5 appear to get darker with each sample because they have higher concentrations of DNA. The band in lane 5 does not appear darker than the band for the previous sample. This could be because the sample was not properly diluted or it may have been caused by a problem with the gel.
09232014- Good job, but remember next time to include more pictures
Weeks 3 & 4 Figure 1: Gel showing results of restriction enzyme digest. Lane 1 contains the 1 kb DNA ladder. Lane 2 contains the uncut plasmid pGBR22. Lane 3 contains the plasmid digested with EcoRI, Lane 4 contains the plasmid digested with PvuII, and Lane 5 is the plasmid digested with both EcoRI and PvuII.
Analysis:
The restriction enzyme digest of pGBR22 was not successful. There are no visible bands in lanes 3-5 which should contain the plasmid cut by the restriction enzymes EcoRI and PvuII. It is possible that the restriction enzyme digest failed because there was not enough plasmid added to the samples, or possibly because the restriction enzymes were not properly deactivated and digested too much of the plasmid.
Weeks 1 & 2
Figure 3: LB and Kanamycin plate with 10 ul DH5 alpha E. coli cells and pNIC-Bsa4 plasmid.
Figure 4: LB and Kanamycin plate with 50 ul DH5 alpha E. coli cells and pNIC-Bsa4 plasmid.
Analysis:
The transformation was unsuccessful because neither of the plates had any growth of bacteria. This could be because the DH5 alpha competent cells were left on ice for 1 hour after the pNIC-Bsa4 plasmid was added instead of thirty minutes. The DH5 alpha cells were also not used within 5 minutes of removing them from the -80 degree freezer, which also could have caused the transformation to fail.
Figure 1: Nanodrop spectrophotometry reading trial 1 for pGBR22 showing the concentration of the plasmid.
Figure 2: Nanodrop spectrophotometry reading trial 2 for pGBR22 showing the concentration of the plasmid.
Thank you Samantha for submitting - Dr. B121714
Virtual Screening:
Figure 7: GOLD binding poses of the top 3 scoring ligands from the NIH clinical collection library (SAM001246783, SAM001246577, and SAM001246653). Poses are shown in PyMOL with 3N28 shown as a surface, and the ligands shown as sticks and colored by element with cyan carbons. Hydrogen bonds between the ligands and active site are represented by black dashes lines.
Analysis:
The vcPSP protein was screened against the NIH clinical collection, the Chembridge 306 library, and the library of in house compounds. Although these are small libraries, a few ligands had high binding scores. However, the two highest scoring ligands did not satisfy Lipinski's rule. The binding poses for the top ligands were analyzed in PyMol. It appears that GOLD did dock ligands in the active site of vcPSP properly. The next step will be to continue virtual screening by screening larger libraries.
Control Ligand Docking:
Figure 1: Active site of 3N28- Protein is shown as a surface and colored by element with green carbons. The active site is colored by element with cyan carbons. The ligand SEP is shown as sticks.
Analysis:
In preparation for virtual screening the active site of the vcPSP protein was defined by aligning it with a homologous protein (PDB ID: 1L7P), extracting the ligand, SEP, from that structure, and then defining the active site around this ligand. The protein was then prepared for virtual screening with MolProbity, and later with Hermes. First, a library of positive and negative control ligands was screened. Based on the GOLD docking scores for these ligands, it appears that the ligands selected were not very good controls. Many of the negative controls scored higher than the positive controls. This could be because the controls chosen were not very good, or it could be due to errors in the way that GOLD docked the ligands.
1242014- Outstanding work
Weeks 11, 12 & 13
Cloning Trial #3:
Analysis:
There was some growth of colonies on the plates, although there also appears to be some contamination. The next step is to make a master plate and grow cultures overnight.
RE Digest:
Analysis:
The pNIC was successfully cut with BsaI restriction enzyme. Then it was cleaned using PCR clean-up to remove contamination.
Cloning, Trials 1 & 2:
Analysis:
Both of these cloning trials were unsuccessful as shown by the lack of colonies growing on the plates. There could have been errors making the pNIC accepting vector or with the transformation.
Secondary PCR and PCR Squared:
Analysis:
Secondary PCR was conducted so that more PCR squared could be made in preparation for cloning. The first to attempts at PCR squared were unsuccessful, likely due to errors with PCR technique.
RE Digest:
Analysis:
pNIC was sucessfully cut with BsaI as shown by the gel. The sacB gene was removed. The pNIC sample is know ready to use in cloning.
PCR Squared:
Analysis:
PCR squared was conducted to amplify the vcPSP gene. Now, enough of the gene has been made to do the cloning procedure.
1162014- Nice work
Weeks 8,9 & 10
PCR Squared:
Analysis:
The purpose of PCR squared was to amplify the gene produced by secondary PCR. I used two different secondary samples for PCR squared, and only one of them was sucessful. The next step will be to redo PCR squared and PCR clean up or possibly gel extraction. After completing PCR my gene will be ready for cloning.
Secondary PCR:
Analysis:
Secondary PCR was attempted using many different thermocycling conditions. However, none of the conditions used worked for secondary PCR. This could have been because the ideal conditions for my protein were not found, or it may have been caused by problems with PCR technique or experimental error. Avery's secondary sample was then used to move on to PCR squared.
9232014- Keep up the good work
Weeks 5,6 & 7
Primary PCR:
Trial 3:
Trial 2:
Figure 5: Gel electrophoresis results for trial 2 of primary PCR for vcPSP oligo mix. Lane 2 contains the 1Kb DNA ladder, lane 3 contains my primary PCR sample with no visible results, and lane 4 contains Cidia's secondary PCR sample.
Trial 1:
Analysis:
The first 2 trials for my primary PCR were unsuccessful and nothing was seen on the gels for those trials. The third trial of primary PCR was successful, and a smear can clearly be seen on the gel. For trial one the NEB recommended guidelines were used for PCR, and for trial 2 the other guidelines were used. For the successful PCR the annealing time was changed from 10 to 20 seconds. This likely allowed more time for the oligos to successfully attach to one another.
Midiprep:
Figure 3: Nanodrop image for Midi Prep sample measured at 260 nm on the DNA-50 setting showing a 40.7 ng/uL yield for pNIC plasmid.
Figure 2: Nanodrop image for Midi Prep sample measured at 260 nm on the DNA-50 setting showing a 40.6 ng/uL yield for pNIC plasmid.
Analysis:
The purpose of Midi Prep was to isolate the pNIC plasmid that we grew from the cell pellet so that only the plasmid DNA remained in solution. Then the sample was nanodropped to determine the concentration of the plasmid. The average yield for my midiprep sample was 40.65 ng/uL, which is approximately what would be expected for pNIC because it is a low copy plasmid.
PCR- pGBR22:
Analysis:
The PCR of the purple protein coding sequence of pGBR22 was successful The bands in lanes 3-5 appear to get darker with each sample because they have higher concentrations of DNA. The band in lane 5 does not appear darker than the band for the previous sample. This could be because the sample was not properly diluted or it may have been caused by a problem with the gel.
09232014- Good job, but remember next time to include more pictures
Weeks 3 & 4
Figure 1: Gel showing results of restriction enzyme digest. Lane 1 contains the 1 kb DNA ladder. Lane 2 contains the uncut plasmid pGBR22. Lane 3 contains the plasmid digested with EcoRI, Lane 4 contains the plasmid digested with PvuII, and Lane 5 is the plasmid digested with both EcoRI and PvuII.
Analysis:
The restriction enzyme digest of pGBR22 was not successful. There are no visible bands in lanes 3-5 which should contain the plasmid cut by the restriction enzymes EcoRI and PvuII.
It is possible that the restriction enzyme digest failed because there was not enough plasmid added to the samples, or possibly because the restriction enzymes were not properly deactivated and digested too much of the plasmid.
Weeks 1 & 2
Analysis:
The transformation was unsuccessful because neither of the plates had any growth of bacteria. This could be because the DH5 alpha competent cells were left on ice for 1 hour after the pNIC-Bsa4 plasmid was added instead of thirty minutes. The DH5 alpha cells were also not used within 5 minutes of removing them from the -80 degree freezer, which also could have caused the transformation to fail.